1. Field of the Invention
The present invention relates to a modified product of a segment of HGF (hepatocyte growth factor) used for preventing and treating cancers or diseases caused by excessive neovascularization. More specifically, the present invention relates to a segment of HGF, modified by deficiency of glycosylation.
2. Description of the Related Art
For therapy of cancers, surgical therapy, chemotherapy, radiation therapy or multidisciplinary therapy combining them and the like are conducted, however, remarkable anticancer methods are not established yet. Though a primary tumor can be removed by surgical therapy, there is often formation of invisible small metastatic cancers when the cancer is found in the case of cancers showing a tendency of metastasis such as pancreatic and lung cancers, even if the size of the primary tumor is small, and surgical removal thereof is difficult. In chemotherapy and radiation therapy using an anticancer agent, it is difficult to prevent recurrence of a resistant cancer and metastasis of survived cancer cells although the primary tumor may be temporarily decreased in size. With an anticancer agent and radiation therapy, killing also normal cells in addition to cancer cells is accompanied by severe side effects, resulting in decrease in the quality of life and immune power of a patient. Thus, there is currently no method of efficiently preventing cancer metastasis, and there is a strong desire for development of drugs effective for preventing cancer metastasis.
There are a lot of researches conducted in the past regarding the mechanisms of cancer metastasis. Many cancers develop in epithelial tissue, and cancer cells are released from a primary tumor, break a basement membrane partitioning epithelial tissue, invade into surrounding tissues, enter blood and lymphatic vessels and are carried to distal tissues by blood and lymph flow, and again manifest invasion into tissues from blood and lymphatic vessels and growth therein associated with neovascularization.
For suppressing cancer metastasis, it is believed advantageous to inhibit any of these processes, and listed as cancer metastasis suppressing agents are, for example, substances suppressing adhesion of cancer cells with vascular endothelial cells at metastasis site (see non-patent literature 1), neovascularization inhibitors (see non-patent literature 2), substances suppressing invasion of cancer cells (see patent literature 1), substances inhibiting enzymes which degrade basement membranes (see patent literature 2, non-patent literature 3) and the like.
Recently, tumor dormacy therapy is attracting attention regarding cancer therapy. The tumor dormacy therapy makes a dormant condition of cancer cells using a neovascularization inhibitor. The neovascularization inhibitor does not directly kill cancer cells but inhibits neovascularization to block a route for feeding oxygen and nutrition necessary for growth of cancer cells, which results in inducing apoptosis and making a dormant condition of cancer cells. Known as the neovascularization inhibitor are Angiostatin (see non-patent literature 2), Endostatin (see non-patent literature 4), and the like.
Neovascularization occurs by proliferation of capillary vessels of preexisting blood vessels. Neovascularization is indispensable for many physiological processes such as embryogenesis, wound healing, tissue and organ regeneration, while abnormal neovascularization occurs under pathogenic conditions such as tumor growth and at metastatic tumor sites. Initiation of tumor neovascularization is known to be induced by vascular endothelial cell growth factor (VEGF), basic fibroblast growth factor (bFGF), HGF (HGF; Hepatocyte growth factor) and the like.
In addition to such a neovascularization action, HGF has a mitogenic activity, motogenic activity and morphogenic activity, and originally works as a regeneration factor supporting natural healing power of a living body. It is known that various biological activities of HGF are expressed through binding of HGF to a c-Met/HGF receptor on a target cell. The c-Met/HGF receptor is known to be often excessively expressed in many cancer cells (see non-patent literatures 5 to 7), and HGF induces invasion and metastasis of tumor cells (see non-patent literature 8). Cancer cells invade and metastasize by utilizing the actions of HGF owned by a living body through mutual interactions with interstitial cells surrounding the cancer cells (see non-patent literatures 8, 9). Therefore, it is supposed that if binding of HGF to a c-Met/HGF receptor on tumor cells is inhibited, invasion and metastasis of the tumor cells can be suppressed.
NK4 is a protein composed of an N-terminal hairpin domain and four kringle domains of an α-chain of HGF. NK4 binds to a c-Met/HGF receptor and acts as an antagonist against HGF (see non-patent literatures 10, 11). NK4 is known to suppress invasion and metastasis of a tumor by an HGF antagonist activity. Further, NK4 is known to suppress neovascularization induced not only by HGF but also by VEGF and bFGF by a mechanism other than an HGF antagonist activity (see non-patent literature 12). Thus, NK4 is a bi-functional molecule having an HGF antagonist activity and a neovascularization inhibitory activity simultaneously, and NK4 is expected to be utilized as a novel anticancer agent.
Further, NK4 can be expected, based on its neovascularization inhibitory activity, to be also utilized as an agent for preventing or treating various diseases ascribable to blood vessel abnormal proliferation, for example, rheumatic arthritis, diabetic retinopathy, immature infant retinophathy, senile macular degeneration, excess scar formation in wound healing and the like. As described above, neovascularization is indispensable for maintaining metabolism of tissues under normal condition and keeping functional homeostasis of a living body, however, it is known that abnormal neovascularization is related with various diseases including inflammatory diseases. For example, it has been reported that metastasis and recurrence of solid tumors and diseases such as proliferative diabetes, psoriasis vulgaris, rheumatic arthritis, diabetic retinopathy, senile macular degeneration, excess scar formation in wound healing are ascribable to abnormal proliferation of blood vessels, particularly, peripheral capillary vessels (see non-patent literatures 13 and 14). NK4 having a neovascularization inhibitory activity is promising as an agent for preventing or treating such diseases.
Further, NK4 can be expected to provide application as a drug for infection prevention and treatment of Listeria and malaria. It is known that a c-Met/HGF receptor is used as a base for human infection with Listeria monocytogenes (see non-patent literature 15). Furthermore, activation of a c-Met/HGF receptor by HGF is known to be essential for the initial mechanism of human infection with malaria parasite (see non-patent literature 16). Therefore, it is believed that NK4, an antagonist against HGF, manifests an effect of prevention and treatment of these infectious diseases.
It is necessary to mass-produce an NK4 protein using cells by genetic engineering methods for use, as a pharmaceutical preparation, of an NK4 protein exerting an effect of prevention and treatment of various diseases, as described above. Conventionally, it is known that NK4 can be produced using animal cells such as Chinese hamster ovary (CHO) cells (see patent literature 3), however, in general, methods of producing a protein using animal cells such as CHO cells are expensive, indicating resultant increase in drug price.
As a method of producing a recombinant protein at low cost, known is a method of introducing the intended gene into a prokaryote such as E. coli and allowing this to express (see non-patent literature 17). However, there is a problem that a sugar chain cannot be added to the recombinant protein produced in a prokaryote such as E. coli. The reason for this is that a prokaryote such as E. coli does not contain an endoplasmic reticulum and Golgi apparatus which are places for biosynthesis of a sugar chain.
Addition of a sugar chain to a protein and its modification in an animal cell is post-translational modification using no template, differing from the case of biosynthesis of DNA or proteins. This post-translational modification is conducted through a complicated mechanism via a lot of glycosylation-related enzymes locally present in cell organellas called endoplasmic reticulum and Golgi apparatus. That is, according to a complicated biosynthetic pathway catalyzed by enzymes (glycosidases and glycosyltransferases) specific to certain linkages of monosaccaharides, a sugar chain is elongated so as to obtain a given structure while being subjected to sequential cutting and addition of monosaccharides (see non-patent literature 18). It is known that sugar chains thus added to proteins are widely involved in whole life phenomenon in higher organisms (see non-patent literatures 19, 20).
It is known that half or more of proteins in a human body are present in the form of a glycoprotein carrying sugar chains (see non-patent literature 21), and if a glycoprotein originally present in the form carrying sugar chains is converted into a form containing no sugar chain, there is a fear of losing activity. For example, it is known that erythropoietin known as an erythropoietic hormone loses pharmaceutical activities when sugar chains are removed (see non-patent literature 22).
Yeast is known as a cell which is a host capable of producing a recombinant protein at low cost and having a glycosylation ability (see non-patent literatures 23 to 25). Since yeast is a eukaryote and has an endoplasmic reticulum and Golgi apparatus, it is consequently equipped with glycosylation mechanism. However, since the glycosylation mechanism of yeast differs significantly from that of animal cells, when a protein having glycosylation site(s) is produced in yeast, sugar chain(s) of yeast type would be added. The sugar chain structures of yeast differ significantly from those of other mammals (see non-patent literature 26), and such a recombinant protein manifests antigenicity against human and other mammals, therefore, it cannot be used as a medicament for human and animals.
Further, an insect cell is also a host having a glycosylation ability and can produce a protein at relatively low cost, however, the sugar chain structures of an insect cell are also different from those of human type (see non-patent literature 27), and there is a possibility for a recombinant protein derived from insect cells to show antigenicity against human and other mammals.
Then, one can envisage production of a protein containing no sugar chains by removal of sugar chains from a protein produced using yeast and insect cells and the like, or by introduction of a gene designed to have mutation(s) at glycosylation sites in a protein molecule into yeast and insect cells and the like. However, if a protein originally present in the form carrying sugar chains is converted into a protein containing no sugar chain, there is a fear of losing activity, as described above.
NK4 is a fragment of an HGF α-chain and contains three glycosylation sites of an HGF α-chain (see non-patent literatures 28 and 29). There is utterly no information regarding whether or not NK4 has an activity when sugar chains are removed from NK4.
(Patent Literature 1)
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